Isolation and Characterization of Achromobacter Sp. CX2 From

Home , Achromobacter, Cellodextrin, Cytophagales

Ann Microbiol (2015) 65:1699–1707 DOI 10.1007/s13213-014-1009-6

ORIGINAL ARTICLE

Isolation and characterization of Achromobacter sp. CX2 from symbiotic Cytophagales, a non-cellulolytic bacterium showing synergism with cellulolytic microbes by producing β-glucosidase

Xiaoyi Chen & Ying Wang & Fan Yang & Yinbo Qu & Xianzhen Li

Received: 27 August 2014 /Accepted: 24 November 2014 /Published online: 10 December 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract A Gram-negative, obligately aerobic, non- degradation by cellulase (Carpita and Gibeaut 1993). There- cellulolytic bacterium was isolated from the cellulolytic asso- fore, efficient degradation is the result of multiple activities ciation of Cytophagales. It exhibits biochemical properties working synergistically to efficiently solubilize crystalline cel- that are consistent with its classification in the genus lulose (Sánchez et al. 2004;Lietal.2009). Most known Achromobacter. Phylogenetic analysis together with the phe- cellulolytic organisms produce multiple cellulases that act syn- notypic characteristics suggest that the isolate could be a novel ergistically on native cellulose (Wilson 2008)aswellaspro- species of the genus Achromobacter and designated as CX2 (= duce some other proteins that enhance cellulose hydrolysis CGMCC 1.12675=CICC 23807). The strain CX2 is the sym- (Wang et al. 2011a, b). Synergistic cooperation of different biotic microbe of Cytophagales and produces β-glucosidase. enzymes is a prerequisite for the efficient degradation of cellu- The results showed that the non-cellulolytic Achromobacter lose (Jalak et al. 2012). Both Trichoderma reesi and Aspergillus sp. CX2 has synergistic activity with cellulolytic microbes by niger were co-cultured to increase the levels of different enzy- producing β-glucosidase. To our knowledge, this is the first matic components (Kumar et al. 2008). However, the ability of report on the synergetic effect of the combination of non- major cellulolytic member of microbial strains identified so far cellulolytic and cellulolytic microbes, which is significant to produced a limiting level of one or other enzymatic component help understand the cellulolytic mechanism of cellulose- (Maki et al. 2009). Thus, search for the potential source of digesting Cytophagales. cellulolytic activity is continuing in the interest of successful bioconversion of lignocellulosic biomass. Keywords Achromobacter . Cellulolytic . β-Glucosidase . In our study of cellulose degradation, it was found that the Cytophagales . Isolation . Synergic cellulolytic Cytophagales were always associated with some non-cellulolytic microbes in their process of cellulose digestion. Kato et al. (2008) also confirmed that the cellulose- degrading culture contains four organisms, but only one can Introduction hydrolyze cellulose, and the others are not directly linked to cellulose hydrolysis. To eliminate those non-cellulolytic con- Cellulose consists of only a linear structure of β-1,4-linked taminants, serial dilution and parallel streaking on the cellu- glucose residues, but is very difficult to be degraded, because lose medium was repeatedly performed. However, even after it is highly crystalline and often tightly intertwined together the cellulolytic Cytophagales were grown as a single colony with hemicelluloses and other polymers presenting a barrier to on the cellulose plate, non-cellulolytic strains were still associated with it, because such an association can develop upon X. Chen : Y. Q u School of Life Science, Shandong University, Jinan 250100, People’s transferring to the glucose agar plate. In order to understand Republic of China the attribution of those associations on cellulose digestion, one such non-cellulolytic microbe was isolated. It was observed : : : * X. Chen Y. Wan g F. Yang X. Li ( ) that the novel isolate could enhance the cellulolytic activity of School of Biological Engineering, Dalian Polytechnic University, Ganjingqu Dalian 116034, People’s Republic of China its partner Cytophagales when co-cultured together, although e-mail: [email protected] it did not show any activity on cellulose in the pure culture. In 1700 Ann Microbiol (2015) 65:1699–1707 this paper, we report on the isolation and some properties of medium used for the taxonomic assay is 0.05 g yeast extract this novel non-cellulolytic association, Achromobacter sp. in 100 mL of the mineral medium. Soaked agar was prepared CX2, and its synergic effect with cellulolytic microbes by by soaking and washing agar gel with distilled water to the produced β-glucosidase. To our knowledge, it is the first remove soluble contaminant after agar was melted in water report on non-cellulolytic microbes working synergistically by heating and cooled down to form agar gel. with cellulolytic strains to hydrolyze cellulose. Morphological properties

Materials and methods The unidentified strain was cultured on the glucose medium plate overnight at 37 °C and the cellular morphology was Strains and culture condition observed by light microscopy. The motility was checked in the hanging-drop mount as described previously (Murray Cytophaga sp. LX-7 was isolated previously in our lab (Li and et al. 1994). Flagella were examined with a transmission Gao 1997) and cultured in the mineral medium in the presence electron microscope (TEM) by negative staining. For TEM, of 0.5 % (w/v) cellulose CF11 (Whatman) and 0.2 % (w/v) a droplet of the diluted culture was fixed with 3 % potassium peptone at 30 °C and 150 rpm. The mineral medium consists phosphotungstate (pH 7.0) and the observation was made on a of (per liter) 0.2 g MgSO4·7H2O, 0.75 g KNO3,0.5g model JEM-2000EX TEM. K2HPO4, 0.02 g FeSO4·7H2O,and0.04gCaCl2·2H2O, pH 7.0–7.2. Penicillium decumbens JU-A10 was obtained Physiological and biochemical characteristics by physical and chemical mutagenesis (Sun et al. 2008)and cultured at 30 °C and 160 rpm in the medium containing (per The Hucker staining method was used for Gram staining liter) 30 g wheat bran, 6 g microcrystalline cellulose, 30 g (Murray et al. 1994). Oxidase activity was detected using corncob residue, 2 g (NH4)2SO4,1gurea,3gKH2PO4,0.5g 1 % tetramethyl-p-phenylene-diamine as substrate, and the MgSO4,0.3gCaCl2, 4 g peptone, and 1 g Tween-80. result was read within 30 s. Catalase activity was determined by mixing 3 % hydrogen peroxide with colonies cultured for Isolation of non-cellulolytic strain 24–48 h, and the formation of gas bubbles within 2 min indicated a positive result. Nitrate reduction was tested on five A soil sample was collected from the surrounding enrichment consecutive days of incubation in culture medium containing with lignocellulose at the Bangcuidao Garden of Dalian, and 0.1 % KNO3 and a negative result was confirmed by adding inoculated on the filter paper agar plate by stamping the soil zinc dust. Urea hydrolysis was observed on Christensen urea sample on the filter paper with the flat end of glass rod. The agar slant and the occurrence of a red-violet color was con- inoculated plates were incubated at 37 °C and the transfers sidered as positive. Hydrolysis of starch, casein, esculin and were made as early as possible from the margin of the gelatin, methyl red (MR) reaction, Voges-Proskauer (VP) test, decomposed filter paper to a fresh filter paper agar plate when indole production, hydrogen sulfide production, and citrate cellulolytic bacteria grew. The transfer was performed by usage were carried out as described elsewhere (Smibert and streaking on the filter paper agar plate, and the plates were Krieg 1994). The assimilation of glucose, xylose, maltose, incubated at 37 °C for 5–6 days. This transfer was carried out sucrose, and cellobiose was determined by incubating the over ten times to set up a steady cellulolytic association. A 10- unidentified isolate in the mineral solution supplemented with fold serial dilution of the bacterial suspension with sterile 0.2 % of each carbohydrate at 37 °C for 7 days. Acid and gas saline was spread on the glucose medium plate, in which the production were tested at the same time as described by bacterial suspension was prepared by mixing cellulolytic bac- Smibert and Krieg (1994). Resistance to sodium chloride teria picked out from the thoroughly digested filter paper with was detected by growing on the glucose medium plate sup- the sterile glucose medium. After incubation at 37 °C over- plemented with 1–6 % NaCl at 0.5 % intervals. night, the individual single colonies were picked out from the glucose medium plates and incubated on the fresh glucose Nitrogen and carbon source usage medium plate. To ensure the strain purity, the isolate was streaked on the glucose medium plate and the single colony Nitrogen sources for cell growth were evaluated by comparing was used for the further study. the turbidity of the culture in mineral solution supplemented Filter paper agar plate was prepared by placing a piece of with 0.3 % of glucose and 0.2 % nitrogen sources including filter paper (Whatman No. 1, 8.5 cm in diameter) on a mineral yeast extract, peptone, beef extract, urea, sodium nitrate, am- medium with 2 % (w/v) soaked agar. Glucose medium con- monium citrate, diammonium phosphate, and ammonium sul- tains (per liter) 15 g glucose, 5 g beef extract, 10 g peptone, fate. To test the carbon sources for cell growth the unidentified and 20 g soaked agar in the mineral medium. The basal strain was incubated in the basal medium supplied with 0.3 % Ann Microbiol (2015) 65:1699–1707 1701 carbon sources including glucose, fructose, maltose, soluble MegAlin program in DNAStar package (DNAStar Inc., Mad- starch, cellobiose, sucrose, lactose, carboxymethyl cellulose, ison, USA). mannose, and inulin. Cellulase preparation Temperature and pH growth Cytophaga sp. LX-7 was cultured in a 250-mL flask contain- The temperature range for cell growth was determined by ing 60 mL of mineral medium in the presence of 0.5 % incubating the unidentified isolate in the glucose medium at cellulose CF11 (Whatman) and 0.2 % peptone at 30 °C and different temperatures ranging from 5 to 60 °C. The pH range 150 rpm for 6 days. The culture supernatant was obtained as for cell growth was evaluated by culturing the unidentified cellulase by centrifugation at 4 °C and 10,000 g for 15 min. isolate in the same medium at pH 2.0–10.0. The optimum After P. decumbens JU-A10 was incubated at 30 °C and temperature or pH for the maximum cell growth was detected 160 rpm for 5 days, the culture supernatant was collected as by comparing the absorbance value of the culture. fungal cellulase by centrifugation at 4 °C and 10,000 g for 15 min. The isolate CX2 was incubated in the glucose medi- Extraction and guanine-plus-cytosine content of genomic um at 37 °C and 150 rpm for 48 h, and the culture supernatant DNA was obtained by centrifugation at 4 °C and 10,000 g for 15 min. Genomic DNA was extracted from the cells growing in the glucose medium at 37 °C overnight and purified using the Enzyme assay protocol described previously (Moore and Dowhan 1997). The guanine-plus-cytosine (G+C) content of DNA was Cellulase activity on Avicel was determined by incubating determined by thermal denaturation (Marmur and Doty 1 mL of the culture supernatant with 1 mL of 2 % Avicel 1962). Sample DNA was dissolved in 0.1×SSC buffer (Serva) in 100 mM phosphate-citrate buffer (pH 6.6) at 37 °C (0.15 M sodium chloride ⁄ 0.015 M sodium citrate buffer, for 2 h. The soluble carbohydrate and reducing sugar was pH 7.0) to make a concentration of OD280 between 0.3 and assayed respectively by phenol-sulfuric acid method (Dubois 0.5. The prepared sample was placed in a thermo-cuvette and et al. 1956) and dinitrosalicylic acid method (Miller 1959) the temperature was increased at a rate of 1 °C/min. Melting after the insoluble material was sedimented by centrifugation. temperature (Tm) was determined at the mid-point of the One unit of cellulase was designated as the amount of enzyme denaturing curve. G+C content was calculated according to that produced 1 μmol glucose equivalent per min under the the following equation: G+C content (%)=(Tm −69.3)× condition as described above. Cellulase activity on filter paper 2.44×100 %. DNA of Escherichia coli K12 was used as a was determined using a 1×6 cm strip of Whatman No. 1 paper standard (G+C=50.6 mol %) and was always assayed togeth- as described previously (Mandels et al. 1976). β-Glucosidase er with the tested DNA to confirm the reliability of the results. was determined by measuring the release of p-nitrophenol as previously reported (Li and Gao 1997). Phylogenetic analysis based on 16S rDNA gene sequence Cellulolytic synergism assay The amplification and sequencing of 16S rDNA gene from a single colony cultured on the glucose medium overnight was To assay the synergy between isolate CX2 and Cytophaga sp. commissioned to Takara Co. (Dalian, China). To avoid mis- LX-7, cellulase activity on filter paper or Avicel in the culture reading as a result of PCR error, the sequencing was repeated supernatant of the isolate CX2 together with strain LX-7 was at least twice. assayed by measuring the soluble carbohydrate (Dubois et al. The 16S rDNA gene sequence of the unidentified strain 1956) and reducing sugar (Miller 1959), respectively. The was compared with those available in the GenBank/EMBL synergistic reaction was performed at 37 °C for 2 h in the database and Ribosomal Database Project-II, and the se- suspension of 1 % Whatman No. 1 filter paper or Avicel quences of the closely related strains were retrieved (Johnson cellulose (Serva) in 50 mM phosphate-citrate buffer et al. 2008;Coleetal.2009). Those sequences were aligned (pH 6.6). The culture of the isolate CX2 and strain LX-7 with CLUSTAL X program and corrected manually (Thomp- was heated at 100 °C for 15 min and used for control to son et al. 1997). Phylogenetic analysis was performed using remove the interference of soluble carbohydrate occurred in the PHYLIP software package by bootstrapping (100 repli- the culture supernatant. cates) with programs SEQBOOT, DNADIST, NEIGHBOR, To determine the synergy between isolate CX2 and and CONSENSE (Felsenstein 2005). The percentage similar- P. decumbens JU-A10, cellulose hydrolysis was performed ities of 16S rDNA gene sequence of the unidentified strain at 50 °C for 8 h and the release of reducing sugar was with other closely relative bacteria were calculated with measured (Miller 1959). The synergistic test system consisted 1702 Ann Microbiol (2015) 65:1699–1707 of 7.5 mg Whatman No. 1 filter paper, 100 μL50mMacetate Phenotypic characterization buffer (pH 4.8), 25 μL JU-A10 culture supernatant, and 25 μL CX2 culture supernatant. For the control test, the culture The strain CX2 was Gram-negative and had straight rods supernatant of strain CX2 and strain JU-A10 was replaced (1.0–2.0×0.5–1.0 μm) with rounded ends. It is motile with with 25 μL acetate buffer, respectively. The inactivated super- peritrichous flagella and obligate aerobe. Colonies on the natant was heated to 100 °C for 15 min and used as a blank to glucose agar are circular, opaque, flat, or slightly convex with exclude the interference of reducing sugar existed in the broth. smooth margin and white in color. All assays were performed in triplicate. Strain CX2 exhibited the enzyme activity in oxidase and catalase, but not in lysine decarboxylase, ornithine decarbox- Production of β-glucosidase ylase, arginine dihydrolase and urease. It produced acid from xylose, maltose, sucrose, and cellobiose, but not from glucose. To evaluate the optimum temperature for β-glucosidase pro- The tests in gas production from glucose and nitrate reduction duction, isolate CX2 was incubated in the glucose medium for were positive, and both MR reaction and VP test were nega- 36 h at different temperatures ranging from 25 to 45 °C, and tive. The isolate CX2 failed to hydrolyze gelatin, starch, and β-glucosidase activity in the culture supernatant was deter- casein, but it did hydrolyze esculin. The carbohydrates capa- mined. The optimal pH for β-glucosidase production was ble of being assimilated included D-glucose, D-xylose, malt- analyzed by culturing the isolate CX2 in the glucose medium ose, and cellodextrin. There was no production of hydrogen with different pH. The effect of carbon source on β- sulfide and indole. Strain CX2 could grow in the mineral glucosidase production was determined by assaying β- medium with glucose as the sole carbon and energy source, glucosidase activity in the culture supernatant after incubating and use citrate for growth. in the mineral medium with 0.2 % (w/v) peptone and 0.5 % of different carbohydrates at 30 °C and 150 rpm for 36 h. Phylogenetic analysis After incubating in the glucose medium overnight, a 2 mL culture of the isolate CX2 was inoculated in 200 mL of fresh The 16S rDNA gene from the isolate CX2 was sequenced and glucose medium and incubated at 37 °C with shaking at a 1475-base sequence was obtained (accession JX645344, 150 rpm. The culture was sampled at 4 h intervals over 36 h GenBank), which was most similar to that of the species incubation. Cell concentration was determined by measuring belonging to the genus Achromobacter. The level of sequence the culture turbidity at 600 nm and β-glucosidase activity in similarities among strain CX2 and species of the genus the culture supernatant was evaluated using the above- Achromobacter ranged from 98.1 to 99.5 %. Strain CX2 mentioned method. exhibited the highest sequence similarities to the sequence of Achromobacter ruhlandii. A dendrogram generated by the Statistical analysis neighbor-joining method was shown in Fig. 1, in which the dataset used for the construction of the phylogenetic tree All of the assays and determinations described in this paper contained 1456 nucleotides. Our strain formed a phylogenetic were performed in triplicate unless otherwise stated. The data cluster with A. ruhlandii. were subjected to one-way analysis of variance (ANOVA) to The GenBank accession numbers were shown in Fig. 1. detect statistical significance. DNA base composition

The G+C content of strain CX2 was 65.1 mol %, which was Results comparable to that of the genus Achromobacter ranging from 65 to 68 mol %. Isolation of associated cellulolytic bacterium Culture condition for cell growth After several transfers by streaking on the filter paper agar plates, a yellowish Cytophagales association was established. Good growth was supported by sugars including glucose, Such an association was streaked on the glucose medium sucrose, maltose, mannose, fructose, and cellobiose, but poor plates and several single colonies were selected. The sequenc- growth was observed when cellulose, inulin, starch, lactose, ing results of 16S rDNA gene indicated that all those se- and carboxymethyl cellulose were used. All the tested nitro- quences were identical. Thus, one single colony was purified gen sources including ammonium citrate, ammonium further by streaking on the glucose medium plate, and the biphosphate, ammonium sulfate, sodium nitrate, urea, beef purified species was designated as CX2 for further study. extract, peptone, and yeast extract supported the isolate CX2 Ann Microbiol (2015) 65:1699–1707 1703

Fig. 1 Neighbour-joining phylogenetic tree showing the position of the isolate CX2T within the genus Achromobacter based on 16S rDNA gene sequence data. GenBank accession numbers were shown in parentheses. Bootstrap values expressed as a percentage of 100 replications were given at the branching points. The scale bar represents 1 % sequence dissimilarity

growth. There was no special growth factor requirement by Production of β-glucosidase by Achromobacter sp. CX2 the isolate CX2. Strain CX2 could grow at the temperature range from 4 to The β-glucosidase activity could be detected in the culture 60 °C, and the optimum temperature was at 37 °C. The pH supernatant of the isolate CX2 in the presence of glucose, range for cell growth was from 5 to 11 and maximum growth sucrose, maltose, mannose, fructose, or cellobiose, which was occurred at pH 7.0–8.0. Strain CX2 was not halophilic, but capable of hydrolyzing cellodextrin into glucose. As shown in was able to grow in the presence of NaCl concentrations up to Fig. 3, the maximal β-glucosidase activity was obtained when 4%. the isolate CX2 was cultured at 35 °C and pH 7.0–8.0. The time course of the isolate CX2 growing in the glucose medium

Role of Achromobacter sp. CX2 in cellulose degradation of Cytophaga sp. LX-7

As shown in Fig. 2, there was no cellulase activity on filter paper or Avicel to be detected in the culture of the isolate CX2. However, the cellulase activity of Cytophaga sp. LX-7 was increased by 18–20 % when the culture of Achromobacter sp. CX2 was combined in the culture supernatant of Cytophaga sp. LX-7 and cellulose degradation was detected by measuring the released soluble carbohydrate. It suggested that a synergism between cellulolytic Cytophaga sp. LX-7 and non-cellulolytic strain CX2 was involved in the degradation of filter paper or Avicel. To confirm such a synergistic degradation in the combination of strain CX2 and Cytophaga sp. LX-7 further, cellulase activity was also assayed by the re- Fig. 2 Synergistic interaction occurred between Achromobacter sp. CX2 and Cytophaga sp. LX-7. The culture supernatant of the isolate CX2 was leased reducing sugar. Almost the same cellulase level as that incubated with strain LX-7 together in filter paper or Avicel suspension by measuring the released soluble carbohydrate was observed for 2 h, in which the cellulase was determined by assaying soluble (Fig. 2), whereas only a little cellulase activity on filter paper carbohydrate (clear column) or reducing sugar (filled column). X-labels or Avicel was detected in the culture supernatant of LX-7/CX2 is for the synergistic results between LX-7 and CX2; LX-7 and CX2 is for the cellulolytic result by LX-7 and CX2 individually. Cytophaga sp. LX-7 by measuring the released reducing Symbol ** at the top of the bars indicates significant differences between sugar. the co-culture of LX7/CX2 and the individual LX7 1704 Ann Microbiol (2015) 65:1699–1707

Fig. 5 Synergistic interaction occurred between Achromobacter sp. CX2 and Penicillium decumbens JU-A10. The culture supernatant of strain CX2 was incubated with strain JU-A10 culture filtrate in filter paper reaction system for 8 h. X-labels A10/CX2 is for the synergistic results between A10 and CX2; A10 and CX2 is for the cellulolytic result by A10 and CX2 individually, and A10+CX2 is the sum of the combined A10 and CX2 Fig. 3 Effect of temperature (▲) and pH (●)onβ-glucosidase production by Achromobacter sp. CX2 cultured in the glucose medium Discussion for cell growth and β-glucosidase production is shown in β Fig. 4. -Glucosidase activity increased progressively with Although Achromobacter spp. is commonly isolated from the the cell growth, and the maximal activity was obtained when human gastrointestinal tract as a causative pathogen of various cell growth approached a plateau. infections (Park et al. 2012), some Achromobacter species are also confirmed to exist in the cellulose-degrading bacterial Cellulolytic synergism between isolate CX2 community by means of 16S rDNA comparison or fatty acid and P. decumbens JU-A10 analysis (Dumova and Kruglov 2009). However, there was no single colony to be isolated from the cellulolytic coenosis so The release of reducing sugar from cellulose degradation was far, as well as the function of those strains was not clarified measured when incubated the culture supernatant of (Dumova and Kruglov 2009;Yangetal.2011; Talia et al. P. decumbens JU-A10 or the combined fungal culture filtrate 2012). Recently an Achromobacter species was found to be with the isolate CX2 culture in filter paper suspension. As always associated with the cellulolytic Cytophagales in our shown in Fig. 5, the reducing sugar released by fungal culture searching for cellulose-degrading microbes. In order to under- filtrate mixed with the cell-free culture of strain CX2 was stand its roles in the cellulose digestion, one such greater than the sum of the amounts released by the individual Achromobacter strain was isolated from the cellulolytic asso- fractions. The cellulolytic activity of P. decumbens JU-A10 ciation and designed as CX2. was enhanced by Achromobacter sp. CX2. The taxonomic properties of isolate CX2 are consistent with the key characteristics of the genus Achromobacter including Gram-negative, straight rod, non-spore forming, motile with peritrichous flagella, obligately aerobic, oxidase- and catalase-positive, reduces nitrate to nitrite (Busse and Auling 2005). Therefore, strain CX2 should be phenotypically clas- sified in the genus Achromobacter. Phylogenetic analysis based on 16S rDNA gene sequence also supported that strain CX2 was a member of the genus Achromobacter. Sequence search on the database revealed that the newly determined sequence was closely related to the sequences from strains belonging to the genus Achromobacter. The levels of sequence similarity between the isolate CX2 and species in the genus Achromobacter were higher than 98.0 %, which were in accordance with the proposal of 95 % 16S rDNA gene sequence similarity as a Fig. 4 Cell growth (●)ofAchromobacter sp. CX2 and process of β-glucosidase production (▲) in the glucose medium at 37 °C with cut-off value for delineating genera (Wagner-Döbler et al. shaking at 150 rpm 2004). Moreover, the G+C content of the genomic DNA also Ann Microbiol (2015) 65:1699–1707 1705 fell into the range of 65–68 mol %, which is just the range of No matter how many times the yellowish Cytophagales G+C content for the genus Achromobacter. were purified by serial dilution or parallel streaking on cellu- As shown in the neighbour-joining phylogenetic tree con- lose plates, some identical white colonies develop when trans- structed based on 16S rDNA sequence (Fig. 1), the isolate ferred to the glucose medium plate. A deliberate search was CX2 was clearly clustered into the clade of the genus made for synergic microbes in the Cytophagales association, Achromobacter, in which the most closely related species and Achromobacter sp. CX2 was obtained. However, the was A. ruhlandii with a similarity of 99.5 %. Alcaligenes strain CX2 is a non-cellulolytic microbe, because it could denitrificans also was clustered with isolate CX2 and showed not grow on the cellulose medium and there was no detectable higher homology (99.2 % similarity), whereas it has been cellulolytic activity in the culture of strain CX2 (Fig. 2). This formally transferred to the genus Achromobacter as suggested that the cellulose-digesting Cytophagales were as- Achromobacter denitrificans (Coenye et al. 2003a). Strain sociated with free-living non-cellulolytic microbes. CX2 exhibited over 98 % 16S rDNA sequence similarity with To ascertain the role of Achromobacter sp. CX2 in the the other species in the genus Achromobacter, whereas with cellulose degradation of Cytophagales, the cellulose- the species in the other related genera, such as Bordetella and degrading activity of Cytophaga sp. LX-7 was assayed in Pigmentiphaga, the similarities of 16S rDNA sequence was thepresenceofAchromobacter sp.CX2.Asshownin all lower than 98 % except Bordetella hinzii and B. avium Fig. 2, the cellulase activity of Cytophaga sp. LX-7 increased having 98.2 % similarity. Such a differentiation at the genus by Achromobacter sp. CX2 when cellulose degradation was level was further supported by biochemical characteristics. evaluated by phenol-sulfuric acid (Dubois et al. 1956), sug- Although the phenotypic characteristics of strain CX2 were gesting that non-cellulolytic strain CX2 had a synergism with consistent with its classification in the genus Achromobacter, cellulolytic Cytophaga sp. LX-7 in cellulose degradation. It is the isolate CX2 could be distinguished from its nearest appar- interesting that the significantly increased cellulase activity ent phylogenetic neighbor Achromobacter spp. in some nota- was observed in the combination of strain CX2 and ble phenotypic traits (Table 1), such as denitrification, assim- Cytophaga sp. LX-7 when cellulase activity was determined ilation of D-glucose and D-xylose, hydrolysis of esculin, H2S by measuring reducing sugar (Fig. 2), although only detect- production, acid production. On the basis of the differential able cellulase activity could be detected in the culture of phenotypic and phylogenetic characteristics, it was confirmed Cytophaga sp. LX-7 by assaying reducing sugar. In fact, the that the isolate CX2 could not be assigned to any previously main cellulolytic products by Cytophaga sp. LX-7 were recognized bacterial species. Thus, we propose that strain cellodextrin, which was supported by the reverse cellulase CX2 represents a new member of the genus Achromobacter, activity of Cytophaga sp. LX-7 by assaying the soluble car- and named Achromobacter sp. CX2. bohydrate and reducing sugar (Li and Gao 1997). It was presumed that the oligosaccharide produced by Cytophaga Table 1 Phenotypic characteristics that distinguish Achromobacter sp. sp. LX-7 was hydrolyzed subsequently by Achromobacter CX2 from other related Achromobacter species enzymes, leading to the increased reducing sugar in the syn- Characteristic 1 2 3 4 5 6 7 ergistic degradation of the combination of Cytophaga sp. LX- 7andAchromobacter sp. CX2. Therefore, β-glucosidase was Denitrification + + + −− −−presumably produced by Achromobacter sp. CX2. Assimilation of In the following experiments, such β-glucosidase activity D-Glucose + + −−−−+ was detected in the culture supernatant of Achromobacter sp. D-Xylose + + −−−−+ CX2, which can hydrolyze cellodextrin into glucose. The β- Esculin hydrolysis + − + −− −−glucosidase activity could be detected in the culture superna-

H2Sproduction −−− −+ −−tant with all tested carbohydrates as carbon sources, suggest- Acid production from ing that β-glucosidase was produced constituently by Glucose − + −−−−−Achromobacter sp. CX2. The optimum temperature for β- Xylose + + −−−−−glucosidase production was at 35 °C and the maximal β- Sucrose + −−ND − ND − glucosidase activity was detected in the culture supernatant Citrate utilization + + + + − ++when the isolate CX2 was incubated at pH 7.0 and 8.0 (Fig. 3). To confirm such synergism of β-glucosidase in cellulose References (Busse and Auling 2005; Coenye et al. 2003a, b;Gomilaetal. degradation, the cellulose-degrading activity of P. decumbens 2011; Papalia et al. 2013) JU-A10 was determined in the presence of Achromobacter sp. Species: 1, Achromobacter sp. CX2; 2, A. xylosoxidans;3, β A. denitrificans;4,A. spanius;5,A. piechaudii;6.A. insolitus;7, CX2. It has been reported that the low secretion of - A. ruhlandii glucosidase results in cellobiose accumulation acting as a Symbol: +, 90 % or more of strains are positive; −, 90 % or more of strains feedback-inhibitor of cellulose depolymerisation (Mfombep are negative; d, 11–89 % of strains are positive; ND, no data available et al. 2013). As shown in Fig. 5, the isolate CX2 could 1706 Ann Microbiol (2015) 65:1699–1707 increase the cellulase activity on filter paper of P. decumbens project: improved alignments and new tools for rRNA analysis. – JU-A10 when mixed with the fungal culture filtrate, although Nucl Acids Res 37:D141 D145 Dubois M, Gilles K, Hamilton JK, Rebirs PA, Smith F (1956) the pure culture of strain CX2 did not have any effect on filter Colorimetric method for determination of sugars and related paper. This result suggested a synergistic interaction occurred substrances. Anal Chem 28:350–356 between P. decumbens JU-A10 and strain CX2. Thus, the Dumova VA, Kruglov YV (2009) A cellulose-decomposing bacterial – culture supplement of the strain CX2 presumably is a com- association. Microbiol 78:234 239 β Felsenstein J (2005) PHYLIP (Phylogeny Inference Package) version 3.6. plement to -glucosidase activity in P. decumbens JU-A10 to Distributed by the author. Department of Genome Sciences, allow cellulolytic enzymes to function more efficiently by University of Washington, Seattle scavenging end product cellobiose (Sun et al. 2008;Liu Gomila M, Tvrzová L, Teshim A, Sedláček I, González-Escalona N, et al. 2012). Zdrahal Z, Sedo O, Gonzalez JF et al (2011) Achromobacter marplatensis sp. nov., isolated from a pentachlorophenol- contaminated soil. Int J Sys Evol Microbiol 61:2231–2237 Jalak J, Kurašin M, Teugjas H, Väljamäe P (2012) Endo-exo synergism in cellulose hydrolysis revisited. 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